This project will carry out basic research on the development of new vectors and delivery systems to target invasive tumor cells in the brain, as well as provide testing of pro-drug activation systems for Project 1 (Chiocca) and expertise on Herpes Simplex Virus (HSV) amplicon vectors for Project 3 (Weissleder). New targeting strategies will involve use of vehicle cells to carry therapeutic genes that have a bystander effect and to produce therapeutic vectors on site. Vehicle cells involve neuroprogenitor cells, which have been shown to migrate within the adult brain towards tumor cells, and primary human cell types, CD34+ cells, fibroblasts and endothelial cells, which can enter into tumors through the vasculature. These cells will be marked to follow their distribution in vivo using imaging technologies developed by Dr. Weissleder and the neuropathologic expertise of Dr. Louis. Cells will also be engineered to carry pro-drug activation genes and tested for therapeutic efficacy and brain tumor and subcutaneous tumor models. Two modes of vector delivery will be explored. The first involves conversion of tumor or vehicle cells to retrovirus packaging cells by infection with an HSV "tribrid" amplicon vector which contains elements of Epstein Barry Virus, to allow episomal maintenance in host cells and retrovirus to produce retrovirus vectors The second involves delivery of replication-conditional HSV in a quiescent state from which it can be activated. by drug administration. This will be done using the piggy-back system in which an HSV mutant defective in an immediate early gene is coupled with an amplicon vector bearing this gene under a tetracycline (silencer/tet-on) regulatory element. Pro-drug activation genes to be tested in these vectors for synergistic interactions include: folypolylglutamate synthatase, which enhances the activation of anti-folates P450 reductase, which potentiates conversion of cyclophosphamide to active metabolites; and carboxylesterase which activates CPT-11, tested in collaboration with Drs. Chiocca and Colvin. These HSV-based cellular targeting strategies and therapeutic gene combinations are designed to be compatible with strategies being developed in this P01 for clinical applications by Drs. Hochberg and Chiocca, as well as with current clinical treatment of brain tumor patients.